Synthesis of hydrophobic taxane derivatives

ABSTRACT

This invention provides a taxane derivative of the formula: ##STR1## wherein a hydrophobic organic moiety is attached to a taxane. R and R 1  is each indepently H or a hydrophobic organic moiety, as long as at least one of R and R 1  is not H. Attachment of a hydrophobic organic moiety to the taxane so as to obtain a taxane derivative generally stabilizes the association of the derivative with a lipid, including a liposomal lipid, carrier in the plasma of animals to which the derivative-carrier association is administered. Also provided herein is a composition containing the taxane derivative and a pharmaceutically acceptable medium; desirably, the medium also contains a lipid carrier, and the derivative is associated with the carrier. Further provided herein is a method of administering taxane derivatives to animals, for example, animals afflicted with cancers.

This is a continuation application of Ser. No. 08/753,650 filed on Nov.27, 1996, now pending which is a continuation of Ser. No. 08/474,888filed on Jun. 7, 1995, now U.S. Pat. No. 5,580,899 which is acontinuation-in-part of Ser. No. 08/369,817, filed Jan. 9, 1995, nowabandoned.

This invention provides a taxane derivative comprising a hydrophobicorganic moiety attached to a taxane, compositions comprising suchcompounds, including lipid carrier-containing compositions, and methodsof administering such compositions to animals, including those afflictedwith cancers.

Taxanes can be anticancer agents, which affect cell growth by blockingcell division. Paclitaxel, for example, is an antimitotic agent whichbinds to tubulin, thereby blocking the disassembly of microtubules andconsequently, inhibiting cell division (Schiff et al., Nature 277:665(1979)). The optimal effect of paclitaxel on polymerization andstabilization of microtubules is seen at concentrations nearstoichiometric equivalence with tubulin dimers (Schiff and Horowitz,Proc. Natl. Acad. Sci. U.S.A. 77(3):1561-1565 (1980)). Paclitaxel hasbeen found to have activity against ovarian and breast cancers, as wellas against malignant melanoma, colon cancer, leukemias and lung cancer(see, e.g., Borman, Chemical & Engineering News, Sep. 2, 1991, pp.11-18; The Pharmacological Basis of Therapeutics *****(Goodman Gilman etal., eds.)******, Pergamon Press, New York (1990), p.1239; Suffness,Antitumor Alkaloids, in: "The Alkaloids, Vol. XXV," Academic Press, Inc.(1985), Chapter 1, pp. 6-18; Rizzo et al., J. Pharm. & Biomed. Anal.8(2):159-164 (1990); and Biotechnology 9:933-938 (October, 1991).

Paclitaxel can be isolated from natural sources, or preparedsynthetically frpm naturally occurring precursors, e.g., baccatin, byattachment of protecting groups to the hydroxyl groups of theseprecursors that are to become the hydroxyl groups of paclitaxel,converting the precursors, and then removing the protecting groups fromthe hydroxyl groups to obtain paclitaxel (see, e.g., WO93110076, int.pub. date May 27, 1993; K. V. Rao, U.S. Pat. No. 5,200,534; R. A.Holton, U.S. Pat. No. 5,015,744; PCT/US92/07990; V. J. Stella and A. E.Mathew, U.S. Pat. No. 4,960,790; K. C. Nicolau, Nature 364 (1993), pp.464-466; Nicolaou, K. C. et al. Nature 367 (1994) pp.630-634; Holton, R.A., et al. J. Am. Chem. Soc. 116 (1994) pp. 1597-1600; WO93/16059, int.pub. date Aug. 19, 1993; EP 528,729, published Feb. 24, 1993; EP522,958, published Jan. 13, 1993; WO91/13053, int. pub. date Sep. 5,1991; EP 414,610, int. pub. date Feb. 27, 1991). The protecting groupsused in some of these synthetic processes are short-chain aliphaticalkyl groups, but are not hydrophobic organic moieties as the term isused herein.

Paclitaxel is highly insoluble in water and aqueous solvents, and iscurrently supplied as an emulsion (TAXOL®, Bristol-Myers Squibb) in apolyoxyethylated derivative of castor oil and ethanol (CremophorEL®).However, administration of this formulation generally entailspremedication with other drugs and a slow infusion of a large volume, toavoid toxicity associated with the Cremophor vehicle. Patients aretherefore generally required to be admitted to hospitals over night.Compositions provided herein comprising a taxane derivative associatedwith a lipid carrier can solve this problem, by providing a formulationin which the taxane remains stably associated with the lipid carrierwhen administered. Stable association with a lipid carrier generallyavoids the toxicity problems encountered with the currently useddelivery system, as well as the need for slow-infusion administration.

SUMMARY OF THE INVENTION

This invention provides a taxane derivative of the formula: ##STR2##wherein: A¹ is H or a group having the formula Q--C(O)NHCH(C₆H₅)CH(OR)C(O)--. Q is C₆ H₅ --, (CH₃)₃ CO-- or (CH₃)CH═C(CH₃)--; A² is Hor CH₃ C(O)--; A³ is H, or OH, A¹ is preferably (C₆ H₅)(O)NHCH(C₆H₅)CH(OR)C(O)--. Preferably, A² is CH₃ C(O)--, and A³ is H that is, thetaxane derivative preferably is a paclitaxel.

When R¹ is H, A¹ is a group having the formula Q--C(O)NHCH(C₆H₅)CH(OR)C(O)--; R is then not H, but rather, is a group having theformula Y¹, Z¹ X¹ or Z¹ D¹. When A¹ is H, or when A¹ is a group havingthe formula Q--C(O)NHCH(C₆ H₅)CH(OR)C(O)-- and R is H, R¹ is then not H;rather, R¹ is then a group having the formula Y², Z² X² or Z² D².Accordingly, at least one hydrophobic organic moiety is attached to thetaxane. Furthermore, two hydrophobic organic moieties can be attached tothe taxane, R then being a group having the formula Y¹, Z¹ X¹ or Z¹ D¹when R¹ is a group having the formula Y², Z² X² or Z² D².

Each of Y¹ and Y² is independently a group having the formula:--C(O)(CH₂)_(a) (CH═CH)_(b) (CH₂)_(c) (CH═CH)_(d) (CH₂)_(e) (CH═CH)_(f)(CH₂)_(g) (CH═CH)_(h) (CH₂)_(i) CH₃. The sum of a+2b+c+2d+e+2f+g+2h+i isequal to an integer from 7 to 22 (refering to the number of carbonatoms); a is zero or an integer from 1 to 22; each of b, d, f and h isindependently zero or 1; c is zero or an integer from 1 to 20; e is zeroor an integer from 1 to 17; g is zero or an integer from 1 to 14; i iszero or an integer from 1 to 11; and a to i can be the same or differentat each occurrence.

Each of Z¹ and Z² is independently a linker of the formula:--C(O)(CH₂)_(j) (CH═CH)_(k) (CH₂)_(l) (CH═CH)_(m) (CH₂)_(n) (CH═CH)_(o)CH₂)_(p) (CH═CH)_(q) (CH₂)_(r) C(O)--. The sum of j+2k+l+2m+n+2o+p+2q+ris equal to an integer from 2 to 22; each of k, m, o and q isindependently zero or 1; j is zero or an integer from 2 to 22; / is zeroor an integer from 1 to 20; n is zero or an integer from 1 to 17; p iszero or an integer from 1 to 14; and r is zero or an integer from 1 to11. Each of j to r can be the same or different at each occurrence.

Each of X¹ and X² is independently a group having the formula: ##STR3##G¹ is --OP(O)₂ OCH₂ CH₂ N(CH₃)₃ (phosphorylcholine), --OP(O)₂ OCH₂ CH₂NH₂, (phosphorylethanolamine) --OP(O)₂ OCH₂ CH(OH)CH₂ OH(phosphorylglycerol), --OP(O)₂ OCH₂ CH(NH₂)CO₂ H (phosphorylserine) orphosphoylinositol.

Each of D¹ and D² is independently a group having the formula: ##STR4##

When R is not H, it is preferably a group having the formula Y¹. Y¹ ispreferably a group having the formula --C(O)(CH₂)_(a) CH₃, and stillmore preferably, is --C(O)(CH₂)₁₀ CH₃ or --C(O)(CH₂)₁₆ CH₃. However, Rcan also be a group having the formula Z¹ X¹. G¹ is then preferablyphosphorylcholine, Z¹ is preferably --C(O)(CH₂)₈ C(O)-- and R ispreferably a group having the formula: ##STR5## wherein Y¹ is preferablya group having the formula --C(O)(CH₂)_(a) CH₃.

R can further be Z¹ D¹. Z¹ is then preferably a group having the formula--C(O)(CH₂)_(l) C(O)--, more preferably, --C(O)(CH₂)₃ C(O)--.

When R¹ is not H, it is preferably Y². More preferably, R¹ is then agroup having the formula --C(O)(CH₂)_(a) CH₃, and still more preferably,--C(O)(CH₂)₁₀ CH₃ or --C(O)(CH₂)₁₆ CH₃. However, R¹ can then also be agroup having the formula Z² X² ; G¹ is then preferably phosphorylcholineand Z² is preferably --C(O)(CH₂)₈ C(O)--. R¹ can further be Z² D² ; Z²is then preferably a group having the formula a group having the formula--C(O)(CH₂)_(j) C(O)--, more preferbly, --C(O)(CH₂)₃ C(O)--.

Hydrophobic organic moieties can be attached to the same taxane at boththe 2' and 7 positions; neither R nor R¹ is then H. R and R¹ can both bethe same moiety, such as groups having the formula --C(O)(CH₂)₁₀ CH₃ or--C(O)(CH₂)₁₆ CH₃, or different moieties, but are preferably the samemoiety.

Also provided herein is a composition comprising a pharmaceuticallyacceptable medium and the taxane derivative of this invention. Themedium preferably comprises a lipid carrier, for example, a fatty acid,lipid, micelle, aggregate, lipoprotein or liposome, associated with thetaxane. Preferably, the lipid carrier is a liposome. The lipid carriercan comprise an additional bioactive agent, that is, a bioactive agentin addition to the taxane derivative. Lipid carriers can also comprise aheadgroup-modified lipid.

Further provided herein is a method of administering a taxane derivativeto an animal, preferably a human. The method of this invention can beused to treat an animal afflicted with a cancer, by administering to theanimal an anticancer effective amount of the derivative. "Anticancereffective" amounts of a taxane derivative are typically at least about0.1 mg of the derivative per kg of body weight of the animal to whichthe derivative is administered; generally, the anticancer effectiveamount of the taxane is from about 0.1 mg per kg of body weight to about1000 mg/kg. Preferably, the anticancer taxane derivative administered isa paclitaxel derivative.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Histograms reflecting association of paclitaxel and paclitaxelhydrophobic organic moiety conjugates with palmitoyloleoyl (POPC)liposomes. X-axis: paclitaxel; 2'-C12 paclitaxel (--C(O)(CH₂)₁₂ CH₃attached to paclitaxel at the 2' position); 7-C12 paclitaxel(--C(O)(CH₂)₁₂ CH₃ attached to paclitaxel at the 7 position) and 2x-C12paclitaxel (--C(O)(CH₂)₁₂ CH₃ attached to paclitaxel at the 2' and 7positions). Y-axis: percentage of paclitaxel associated with liposomes.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a taxane derivative of the formula: ##STR6##comprising a hydrophobic organic moiety attached to a taxane. A¹ is H ora group having the formula Q--C(O)NHCH(C₆ H₅)CH(OR)C(O)--. Q is C₆ H₅ -,(CH₃)₃ CO--, or CH₃ CH═C(CH₃)--; A² is H or CH₃ C(O)--; A³ is H or OH.

A¹ is preferably a group having the formula Q--C(O)NHCH(C₆H₅)CH(OR)C(O)--; Q is then preferably C₆ H₅, A² is then preferably CH₃C(O)-- and A³ is then preferably H. Accordingly, paclitaxel ( CompoundI!; TAXOL® (C₄₇ H₅₁ NO), Bristol-Myers Squibb) is preferred herein.However, taxotere (II), which differs from paclitaxel by having atert-butoxy carbonyl group at the C-13 position, instead of a benzoylgroup, and a hydroxyl group, instead of an acetyloxy group, at C-10 isalso useful herein. Accordingly, for taxotere, A¹ is (CH₃)₃COC(O)NHCH(C₆ H₅)CH(OR)(O)--, A² is H, and A³ is H. Cephalomannine(III), differs from paclitaxel in the amide group located at the distalend of the C-13 ester. A¹ is then (CH₃)CH═C(CH₃)C(O)NHCH(C₆H₅)CH(OR)C(O)--, A² is CH₃ C(O)-- and A³ is H. Additional taxanes usefulin accordance with the practice of this invention include, withoutlimitation: 19-hydroxybaccatin III IV!, Baccatin V V!, 10-deacetylcephalomannine VI!, 10-deacetyl paclitaxel VII!, 7-Epi-10-deacetylpaclitaxel VIII!, 7-Epi-10-deacetyl cephalomannine IX!, and 10-deacetylbaccatin III X!, as described in the following table, in addition topaclitaxel, taxotere and cephalomannine. The compound names listed arefor unsubstituted, or "free", taxanes, that is, taxanes to whichhydrophobic organic moieties are not attached.

    ______________________________________    Compound A.sup.1            A.sup.2  A.sup.3    ______________________________________    Paclitaxel             C.sub.6 H.sub.5 C(O)NHCH(C.sub.6 H.sub.5)                                CH.sub.3 C(O)-                                         H    (I)      CH(OR)C(O)-    Taxotere C(CH.sub.3).sub.3 OC(O)NHCH                                H        H    (II)     (C.sub.6 H.sub.5)CH(OR)C(O)-    Cephalo- (CH.sub.3)CH═C(CH.sub.3)C(O)NHCH                                CH.sub.3 C(O)-                                         H    mannine  (C.sub.6 H.sub.5) CH(OR)C(O)-    (III)    19-hydroxy             H                  CH.sub.3 C(O)-                                         OH    baccatin    III    (IV)    Baccatin III             H                  CH.sub.3 C(O)-                                         H    (V)    10-Deacetyl             (CH.sub.3)CH═C(CH.sub.3)C(O)                                H        H    cephalo  NHCH(C.sub.6 H.sub.5)CH(OR) C(O)-    mannine    (VI)    10-Deacetyl             C.sub.6 H.sub.5 C(O)NHCH(C.sub.6 H.sub.5)                                H        H    taxol    CH(OR)C(O)-    (VII)    (7α-OH)    7-Epi-10-             C.sub.6 H.sub.5 C(O)NHCH(C.sub.6 H.sub.5)                                H        H    deacetyl CH(OR)C(O)-    taxol(7β-OH)    (VIII)    7-Epi-10-             (CH.sub.3)CH═C(CH.sub.3)C(O)                                H        H    deacetyl NHCH(C.sub.6 H.sub.5)CH(OR) C(O)-    cephalo    mannine(7β-    OH)    (IX)    10-Deacetyl             H                  H        H    baccatin III    (X)    ______________________________________

R and R¹ can each independently be either H or a hydrophobic organicmoiety, as long as at least one of R and R¹ is not H. "Hydrophobicorganic moieties" are carbon-based molecular groups which can beattached to taxanes. Free taxanes can readily dissociate from lipidswith which they have been associated in the plasma of animals to whichthe taxane/lipid associations have been administered. Attachment of ahydrophobic organic moiety to a free taxane so as to obtain a taxanederivative can stabilize the association of the derivative with a lipid.

Hydrophobic organic moieties include, without limitation, saturated orunsaturated, aliphatic or branched fatty acids. Such moieties alsoinclude: polyol-, e.g., glycerol or mannitol, based amphipathic lipidscomprising a polar group and one or more fatty acids. Furthermore, otherhydrophobic organic moieties, including sphingolipids such assphingomyelin, which can stabilize the association between a taxanederivative and a lipid in an animal's plasma can also be attached to ataxane according to the practice of this invention; selection of suchother moieties is within the purview of ordinarily skilled artisansgiven the teachings of this invention.

"Attachment" means conjugation, covalent binding, linking, conjugationor otherwise forming a chemical connection between a taxane and ahydrophobic organic moiety. Attachment of the moiety is to one or morereactive groups, typically hydroxyl groups, on the taxane. Paclitaxel,for example, has three hydroxyl groups to which hydrophobic organicmoieties can be attached. These are located at the 2', 7 and 1positions, with their relative order of reactivity generally believed tobe (from most reactive to least reactive) 2'>7>>1. Hydrophobic organicmoieties can be attached to the primary reactive group of a taxane,e.g., the 2' OH group of paclitaxel, utilizing stoichiometric amounts ofthe moiety to be attached, e.g., fatty acid chlorides or anhydrides.Reactions are typically performed in the presence of a base, such aspyridine, dimethylaminopyridine, triethylamine, or others, and incommonly used polar, aprotic organic solvents. The progress of thereaction, at room temperature, can be monitored by a number of wellknown chromatographic means, for example, thin layer chromatographyusing a 3% methanol-in-chloroform solvent system. The compound'sidentity can be confirmed by spectroscopic and other analyticalprocedures, such as NMR spectroscopy.

Specific reaction and purification conditions are generally expected tovary according to a number of factors, including without limitation, theraw materials and reactants used, that are well within the purview ofordinarily skilled artisans to determine and control given the teachingsof this invention. For example, for the attachment of lauric acid (C12)to paclitaxel, 9 mg (0.074 mmoles) dimethylaminopyridine (DMAP), 50 mg(0.059 mmole) of paclitaxel and 15 mg (0.068 mmoles) lauroyl chloridecan be combined with 5 ml of chloroform.

Attaching hydrophobic organic moieties to less reactive groups on thetaxane typically requires use of an amount of an active form of themoiety that is in excess of the stoichiometric amount. The hydroxylgroup at the 7 position of paclitaxel, for example, can be modified, forexample, by attaching a hydrophobic organic moiety to both the 2' and 7OH groups, and then selectively removing the 2' moiety, such that themoiety at the 7 position remains attached to paclitaxel. Such reactionscan be performed using essentially the same procedures as thosedescribed above. Selective removal of the 2' modification can beaccomplished using stoichiometric amounts of a mild base, e.g., sodiumbicarbonate.

Additionally, the 7 OH group of paclitaxel can be modified by"protecting" the 2' OH group before covalently linking the drug with thehydrophobic organic moiety. The 2' OH group can also be protected withgroups such as, for example, triphenyl methyl, methoxytriphenyl methyl,trifluoroacetyl and hexanoyl groups, using processes generally known toordinarily skilled artisans. The protected paclitaxel is then reactedwith an active form of the moiety, e.g., fatty acid anhydrides orchlorides, in anhydrous organic solvent and bases such as DMAP andpyridine. The protecting group can be removed from the 2' position bywell known and readily practiced means, under mildly acidic or basicconditions. Lauric acid can, for example, be attached to the 7 OH groupof paclitaxel by combining 54 mg (0.44 mmoles) DMAP, 50 mg (0.059mmoles) paclitaxel, and 77 mg (0.35 mmoles) of lauroyl chloride with 5ml of chloroform, keeping the reaction at room temperature, so as toobtain 2'7-dilauroyl paclitaxel. Then, 3.0 mg NaCl in 75 microliters ofwater can be added to a solution of chloroform/methanol (1:1) containing58 mg (0.048 mmoles) of 2'7-dilauroyl paclitaxel to remove the lauricacid attached to the 2'--OH group. This reaction can be incubated at 30degrees C. and followed closely by thin layer chromatography (TLC).Attachment, however, is not limited to use of these specific amounts;rather, ordinarily skilled artisans can vary the amounts for reasons,and within ranges, well known to them, given the teachings of thisinvention.

R can be H or a group having the formula Y¹, Z¹ X¹ or Z¹ D¹ ; R¹ can beH, or a group having the formula Y², Z² X² or Z² D². At least one of Rand R¹ is not H. Y¹, Z¹ X¹, Z¹ D¹, Y², Z² X² and Z² D² are hydrophobicorganic moieties.

Each of Y¹ and Y² is independently a group having the formula:--C(O)(CH₂)_(a) (CH═CH)_(b) (CH₂)_(c) (CH═CH)_(d) (CH₂)_(e) (CH═CH)_(f)(CH₂)_(g) (CH═CH)_(h) (CH₂)_(j) CH₃. The sum of a+2b+c+2d+e+2f+g+2h+i isequal to an integer from 7 to 22 (refering to the number of carbonatoms); a is zero or an integer from 1 to 22; each of b, d, f and h isindependently zero or 1; c is zero or an integer from 1 to 20; e is zeroor an integer from 1 to 17; g is zero or an integer from 1 to 14; i iszero or an integer from 1 to 11; and a to i can be the same or differentat each occurrence. Preferably, each of Y¹ and Y² is independentlysaturated, that is, there are no double bonds between adjacent carbonatoms. Accordingly, b, d, f and h are each preferably zero, c, e, g, andi are each also zero, and Y¹ and Y² are each independently groups havingthe formula --C(O)(CH₂)_(a) CH₃, wherein a is an integer from 7 to 22.More preferably, each of Y¹ and Y² is independently --C(O)(CH₂)₁₀ CH₃ or--C(O)(CH₂)₁₆ CH₃. Alternatively, Y¹ and Y² can each be unsaturated,that is, they can have one or more CH═CH groups. In this case, at leastone of b, d, f or h is not zero. For example, when the unsaturatedhydrocarbon has one double bond: b is 1, d, f and h being zero; Y¹ andY² are each then independently a group having the formula--C(O)(CH₂)_(a) CH═CH(CH₂)_(c) CH₃ ; a is zero or an integer from 1 to18; c is also zero or an integer from 1 to 18, at least one of a or c isnot zero, and the sum of a and c is equal to an integer of from 5 to 20.

X¹ and X² are each independently a group having the formula: ##STR7## G¹is preferably a phosphate-based polar group, including withoutlimitation: --OP(O)₂ OCH₂ CH₂ N(CH₃)₃ (phosphorylcholine), --OP(0)₂ OCH₂CH₂ NH₂ (phosphorylethanolamine) --OP(O)₂ OCH₂ CH(OH)CH₂ OH(phosphorylglycerol), --OP(O)₂ 0CH₂ CH(NH₂) CO₂ H (phosphorylserine) andphosphorylinositol. More preferably, G¹ is phosphorylcholine. However,nitrogen, sulfur and other atoms can be substituted for the phosphorous.Y¹. is preferably a group having the formula --C(O)(CH₂)_(a) CH₃.

Each of Z¹ and Z² is independently a linker of the formula:--C(O)(CH₂)_(j) CH═CH)_(k) (CH₂)_(l) (CH═CH)_(m) (CH₂)_(n) (CH═CH)_(o)CH₂)_(p) (CH═CH)_(q) (CH₂)_(r) C(O)--. The sum of j+2k+l+2m+n+20+p+2q+ris equal to an integer of from 2to 22; each of k, m, o and q isindependently zero or 1; j is zero or an integer from 2 to 22; l is zeroor an integer from 1 to 20; n is zero or an integer from 1 to 17; p iszero or an integer from 1 to 14; and r is zero or an integer from 1 to11. Each of j to r can be the same or different at each occurrence.Preferably, each of Z¹ and Z² is independently a group having theformula --C(O)(CH₂)_(l) C(O)--, more preferably, each of Z¹ and Z² is--C(O)(CH₂)₈ C(O)--. ##STR8## Y¹ and Y² are then preferably andindependently each a group having the formula --C(O)(CH₂)_(a) CH₃. Forexample, both Y¹ and Y² can each be --C(O)(CH₂)₁₄ CH₃.

When R¹ is H, A¹ is a group having the formula Q--C(O)NHCH(C₆H₅)CH(OR)C(O)--, and R is not H. R is then a group having the formulaY¹, Z¹ X¹, or Z¹ D¹, and the taxane derivative comprises a hydrophobicorganic moiety attached at the 2' position of the taxane. When A¹ is H,or when A¹ is a group having the formula Q--C(O)NHCH(C₆ H₅)CH(OR)C(O--and R is H, then R¹ is not H. R¹ is then Y², Z² X² or Z² D², and thetaxane derivative has a hydrophobic organic moiety attached at the 7position. Alternatively, the taxane derivative can have a hydrophobicorganic moiety attached at both the 2' and the 7 positions of thetaxane; these moieties can be the same or different at each occurence,but are preferably the same. R is then a group having the formula Y¹, Z¹X¹, or Z¹ D¹ when R¹ is a group having the formula Y², Z² X², or Z² D².

Also provided herein is a composition comprising the taxane derivativeof this invention and a pharmaceutically acceptable medium; such amedium preferably comprises a lipid carrier associated with the taxanederivative. "Pharmaceutically acceptable media" are generally intendedfor use in connection with the administration of active ingredients toanimals, for example, humans, and include solids, such as pills,capsules and tablets, gels, excipients or aqueous or nonaqueoussolutions. Active ingredients can, for example, be combined with,dissolved, suspended, or emulsified in or with such media.Pharmaceutically acceptable media are generally formulated according toa number of factors well within the purview of the ordinarily skilledartisan to determine and account for, including without limitation: theparticular active ingredient used, its concentration, stability andintended bioavailability, the disease; disorder or condition beingtreated with the composition; the subject, its age, size and generalcondition; and the composition's intended route of administration, e.g.,nasal, oral, ophthalmic, topical, transdermal, vaginal, subcutaneous,intramammary, intraperitoneal, intravenous, or intramuscular (see, forexample, J. G. Naim, in: Remington's Pharmaceutical Science (A. Gennaro,ed.), Mack Publishing Co., Easton, Pa., (1985), pp. 1492-1517). Typicalpharmaceutically acceptable media used in parenteral drug administrationinclude, for example, D5W, an aqueous solution containing 5% weight byvolume of dextrose, and physiological saline. Pharmaceuticallyacceptable media can contain additional ingredients which enhance thestability of the active ingredients, including preservatives andanti-oxidants.

A "lipid carrier" is a a hydrophobic substance, or an amphipoathicsubstance having a hydrophobic domain, with which the taxane derivativeof this invention can form a stable association, and which is suitablefor therapeutic administration to animals. "Association" as used hereingenerally means association between the hydrophobic organic moietyattached to the taxane and the hydrophobic portion of the lipid carrier.Hydrophobic organic moieties and hydrophobic lipid domains generallyassociate through the action of a number of forces, such as Van derWaal's forces, generally known to operate between hydrophobic moleculesin an aqueous environment. Means of determining the stability of suchassociations, for example, by determining the percentage of taxanederivative recoverable with phosphorous when the lipid carrier comprisesa phospholipid, are well known to, and readily practiced by, ordinarilyskilled artisans given the teachings of this invention. Ordinarilyskilled artisans can, given the teachings of this invention, selectsuitable lipid carriers. These include, without limitation: fatty acids,amphipathic lipids, liposomal or nonliposomal, lipoproteins and others.Preferably, the lipid carrier with which the taxane derivative of thisinvention is associated is a liposome.

Liposomes comprise one or more bilayers of lipid molecules, each bilayerencompassing an aqueous compartment. Unilamellar liposomes have a singlelipid bilayer and multilamellar liposomes have more than one bilayer(for a review see, for example, see Chapman, "Physicochemical Propertiesof Phospholipids and Lipid-Water Systems," in: Liposome Technology,Volume I: Preparation of Liposomes (G. Gregoriadis. ed.). CRC Press,Boca Raton, Fla. (1984). pp. 1-18, the contents of which areincorporated herein by reference). The amphipathic lipid molecules whichmake up lipid bilayers comprise a polar (hydrophilic) headgroup and oneor two acyl chains. The polar groups can be phosphate-, sulfate ornitrogen-based groups, but are preferably phosphate groups, such asphosphorylcholine, phosphorylethanolamine, phosphorylserine,phosphorylglycerol or phosphorylinositiol. The fatty acids generallycomprise from 4 to 24 carbon atoms, and can be saturated (e.g.,myristic, lauric, palmitic, or stearic acids, or unsaturated (e.g.,oleic, linolenic, linoleic and arachidonic acid). Furthermore, liposomescan also comprise sterols, such as cholesterol, and other lipids.

Liposomes can be made by a variety of methods, including: Bangham'smethods for making muiltilamellar liposomes (MLVs); Lenk's, Fountain'sand Cullis' methods for making MLVs with substantially equalinterlamellar solute distribution; extrusion, sonication orhomogenization of MLVs to make unilamellar liposomes; and ether orethanol injection processes (see, for example, U.S. Pat. Nos. 4,522,803,4,588,578, 5,030,453, 5,169,637 and 4,975,282, and R. Deamer and P.Uster, "Liposome Preparation: Methods and Mechanisms," in Liposomes (M.Ostro, ed.), Marcel Dekker, Inc., New York (1983), pp. 27-52, thecontents of which are incorporated herein by reference).

Lipid carriers associated with the taxane derivative of this invention,for example, liposomes, can comprise an additional bioactive agent, thatis, a bioactive agent in addition to the taxane derivative. Liposomes,for example, can be loaded with biologically active agents bysolubilizing the agent in the lipid or aqueous phase used to prepare theliposomes. Alternatively, ionizable bioactive agents can be loaded intoliposomes by first forming the liposomes, establishing anelectrochemical potential, e.g., by way of a pH gradient, across theoutermost liposomal bilayer, and then adding the ionizable agent to theaqueous medium external to the liposome (see Bally et al. U.S. Pat. No.5,077,056, and U.S. Ser. No. 08/112,875, the contents of which areincorporated herein by reference).

Lipid carrier/bioactive agent formulations can enhance the therapeuticindex of the bioactive agent, for example by buffering the agent'stoxicity and by reducing the rate at which the agent is cleared from thecirculation of animals, thereby meaning that less of the agent need beadministered to achieve the desired therapeutic effect. In this regard,lipid carriers, for example, liposomes, can also comprise one or moreheadgroup-modified lipids, which are amphipathic lipids whose polarheadgroups have been derivatized by attachment thereto of a chemicalmoiety, e.g., polyethylene glycol, a polyalkyl ether, a ganglioside, anorganic dicarboxylic acid or the like, which can inhibit the binding ofserum proteins to lipid carriers such that the pharmacokinetic behaviorof the carriers in the circulatory systems of animals is altered (see,e.g., Blume et al., Biochim. Biophys. Acta. 1149:180 (1993); Gabizon etal., Pharm. Res. 10(5):703 (1993); Park et al. Biochim. Biophys Acta.1108:257 (1992); Woodle et al., U.S. Pat. No. 5,013,556; Allen et al.,U.S. Pat. Nos. 4,837,028 and 4,920,016; U.S. Ser. No.142,691, filed Oct.25, 1993). Lipid carriers are generally cleared from animals'circulations by their reticuloendothelial systems (RES). Avoiding RESclearance can allow the carriers to remain in the circulation longer,meaning that less of the associated drug need be administered to achievedesired serum levels. Enhanced circulation times can also allowtargeting of liposomes to non-RES containing tissues. The hydrophobicorganic moiety attached to the taxane can also be a headgroup-modifiedlipid.

The amount of the headgroup-modified lipid incorporated into the carrierdepends upon a number of factors well known to the ordinarily skilledartisan, or within his purview to determine without undueexperimentation. These include, but are not limited to: the type oflipid and the type of headgroup modification; the type and size of thecarrier; and the intended therapeutic use of the formulation. Theconcentration of the headgroup-modified lipid in the carrier isgenerally sufficient to prolong the circulatory half-life of the carrierin an animal, but is not so great as induce unwanted side effects in theanimal, and is typically at least about five mole percent of the lipidpresent in the carrier, The preferred headgroup-modified lipid isdipalmitoyl phosphatidylethanolamine-glutaric acid (DPPE-GA), which istypically used at a concentration of about 10 mole percent of the lipidpresent.

"Bioactive agent" as used herein denotes any compound or composition ofmatter which can be administered to animals and which can havebiological or diagnostic actiovity therein. Bioactive agents include,but are not limited to: antiviral agents such as acyclovir, zidovudineand the intereferons; antibacterial agents such as aminoglycosides,cephalosporins and tetracyclines; antifungal agents such as polyeneantibiotics, imidazoles and triazoles; antimetabolic agents such asfolic acid, purine and pyrimidine analogs; antineoplastic agents such asthe anthracycline antibiotics and plant alkaloids; such as cholesterol;carbohydrates, e.g., sugars and starches, amino acids, peptides,proteins such as cell receptor proteins, immunoglobulins, enzymes,hormones, neurotransmitters and glycoproteins; dyes; radiolabels such asradioisotopes and radioisotope-labelled taxanes; radiopaque taxanes;fluorescent taxanes; mydriatic taxanes; bronchodilators; localanesthetics; and the like. The additional bioactive agent used herein ispreferably an antimicrobial or antineoplastic agent. The additionalbioactive agent can be a therapeutic lipid, such as a ceramide. Theadditional agent can also be a second taxane derivative.

Further provided herein is a method of administering a taxane derivativeto an animal, e.g., a human. The method comprises administering to theanimal a composition comprising the derivative and a pharmaceuticallyacceptable medium. The medium preferably comprises a lipid carrier, morepreferably a liposome, associated with the taxane derivative. The taxanederivative used in the method of this invention comprises a hydrophobicorganic moiety attached to a taxane, and has the formula: ##STR9## A¹ isH or a group having the formula Q--C(O)NHCH(C₆ H₅)CH(OR)C(O)--; Q is C₆H₅ --, (CH₃)₃ C--O-- or (CH₃)CH═C(CH₃)--; A² is H or CH₃ C(O)--; A³ is Hor OH; R is H, or a group having the formula Y¹, Z¹ X¹, or Z¹ D¹ ; andR¹ is H, or a group having the formula Y², Z² X², or Z² D². When R' isH, A¹ is a group having the formula Q--C(O)NHCH(C₆ H₅)CH(OR)C(O)-- and Ris not H; when A¹ is H or when A¹ is a group having the formulaQ--C(O)NHCH(C₆ H₅)CH(OR)C(O)--R¹ is not H; and, at least one of R and R¹is not H. When R¹ is H, R is preferably a group having the formula Y¹.When A¹ is H, or when R is H, R¹ is preferably a group having theformula Y².

Each of Y¹ and Y² is independently a group having the formulaC(O)(CH2)_(a) (CH═CH)_(b) (CH₂)_(c) (CH═CH)_(d) (CH₂)_(e) (CH═CH)_(f)(CH₂)_(g) (CH═CH)_(h) (CH₂)_(i) CH₃. The sum of a+2b+c+2d+e+2f+g+2h+i isequal to an integer of from 2 to 22; a is zero or an integer from 1 to22; each of b, d, f and h is independently zero or 1; c is zero or aninteger from 1 to 20; e is zero or an integer from 1 to 17; g is zero oran integer from 1 to 14; i is zero or an integer from 1 to 11; and a toi can be the same or different at each occurrence. Each of Y¹ and Y² ispreferably, and independently, a group having the formula C(O)(CH₂)_(a)CH₃ --. When R is a group having the formula Y¹, and when R¹ is a grouphaving the formula Y², each is independently preferably --C(O)(CH₂)₁₀CH₃ or C(O)(CH₂)₁₆ H₃.

Each of X¹ and X² is independently a group having the formula: ##STR10##G¹ is --OP(O)₂ OCH₂ CH₂ N(CH₃)₃, --OP(O)₂ OCH₂ CH₂ NH₂, --OP(O)₂ OCH₂CH(OH)CH₂ OH, or --OP(O)₂ OCH₂ CH(NH₂)CO₂ H.

Each of Z¹ and Z² is independently a linker of the formula:--C(O)(CH₂)_(f) (CH═)_(k) (CH₂)_(l) (CH═CH)_(m) (CH₂)_(n) (CH═CH)_(o)CH₂)_(p) (CH═CH)_(q) (CH₂)_(r) C(O)--. The sum of j+2k+l+2m+n+2o+p+2q+ris equal to an integer from 2 to 22; each of k, m, o and q isindependently zero or 1; j is zero or an integer from 2 to 22; l is zeroor an integer from 1 to 20; n is zero or an integer from 1 to 17; p iszero or an integer from 1 to 14; r is zero or an integer from 1 to 11and each of j to r can be the same or different at each occurrence.Preferably, each of Z¹ and Z² independently has the formula:--C(O)(CH₂)_(l) C(O)--; more preferably, each of Z¹ and Z² is--C(O)(CH₂)₈ C(O)--.

Each of D¹ and D² is independently a group having the formula: ##STR11##Preferably, each of Y¹ and Y² is then independently a group having theformula --C(O)(CH₂)_(a) CH₃, for example, --C(O)(CH₂)₁₄ CH₃.

Animals afflicted with cancers can be treated according to the method ofthis invention, by administration of an anticancer effective amount of ataxane derivative provided herein. Paclitaxel derivatives are preferredfor use herein. Generally, those cancers treatable by the method of thisinvention are those which may be treated with the corresponding free,i.e., unattached taxane, and include, without limitation: carcinomas,myelomas, neuroblastomas, or sarcomas, of the brain, breast, lung,colon, prostate or ovaries, as well as leukemias or lymphomas.

Anticancer activity of taxane derivatives can be examined in vitro, forexample, by incubating a cancer cell culture with the derivative, andthen evaluating cell growth inhibition in the culture. Suitable cellsfor such testing include murine P388 leukemia, B16 melanoma and Lewislung cancer cells, as well as human mammary MCF7, ovarian OVCAR-3 andA549 lung cancer cells. GI₅₀ values, that is, the concentration of ataxane derivative required to induce 50% cell growth inhibition in aculture, for a derivative can be determined and compared. The lower ataxane derivative's GI₅₀, the lower is the amount of the derivativerequired to inhibit cancer cell growth. Accordingly, compounds withlower GI₅₀ 's can have better therapeutic indices.

Alternatively, a taxane derivative can be tested in vivo for antitumoractivity, for example, by first establishing tumors in suitable testanimals, e.g., nude mice. Cells suitable for establishing tumors includethose described above for in vitro testing, as well as other cellsgenerally accepted in the art for establishing tumors. Subsequently, thetaxane derivative is administered to the animal; ED₅₀ values, that is,the amount of the derivative required to achieve 50% inhibition of tumorgrowth in the animal are then determined, as are survival rates.Ordinarily skilled artisans, given the teachings of this invention, arewell able to select particular taxane derivatives for applicationagainst certain cancers, on the basis of such factors as GI₅₀, ED₅₀ andsurvival values.

For the purposes of this invention, an "anticancer effective amount" ofa taxane derivative is any amount of the derivative effective toameliorate, lessen, inhibit or prevent the establishment, growth,metastasis, invasion or spread of a cancer. Anticancer effective amountsof taxane derivatives of this invention can be the same amount astherapeutic doses of the corresponding free taxane. However, theattachment of a hydrophobic organic moiety to the taxane so as to obtaina taxane derivative, and the association of this derivative with alipid, e.g., a liposome, in a carrier, can enhance the drug'stherapeutic index. Accordingly, this can mean that less of the taxanederivative, in comparison to the free taxane, need be used to achievethe desired therapeutic effect, and accordingly, that anticancereffective amounts of the derivative can be less than anticancereffective amounts of the free taxane.

Anticancer effective amounts of taxane derivatives can be chosen inaccordance with a number of factors, e.g.; the age, size and generalcondition of the subject, the cancer being treated and the intendedroute of administration of the derivative, and determined by a varietyof means, for example, dose ranging trials, well known to, and readilypracticed by, ordinarily skilled artisans given the teachings of thisinvention. Generally, the anticancer effective amount of the taxanederivative of this invention is at least about 0.1 mg of the derivativeper kg of body weight of the animal to which the composition isadministered. Preferably, the anticancer effective amount of the taxanederivative is from about 0.1 mg per kg to about 1000 mg per kg.

Furthermore, the method provided herein can comprise administering anadditional bioactive agent, typically an antineoplastic agent, to theanimal. This additional bioactive agent can be administered to an animalprior to, concurrently with or subsequently to administration of thetaxane derivative of this invention. The additional agent can beentrapped in a liposome, for example, the same liposome with which thetaxane derivative of this invention can be associated.

This invention will be better understood from the following Examples.However, those of ordinary skill in the art will readily understand thatthese examples are merely illustrative of the invention as defined inthe claims which follow thereafter.

EXAMPLES Example 1

Synthesis of Taxanes

2' caproyl-paclitaxel

Paclitaxel (100 mg, 0.117 mmoles), dimethylaminopyridine (DMAP), (18 mg,0.133 mmoles), and 10 ml of chloroform were combined in an oven-dried,50-ml round-bottom flask with 18 mg (0.147 mmoles) of caproyl chloride,and incubated at room temperature. Reaction progress was monitored bysilica-based thin layer chromatography (TLC), using a 3%methanol-in-chloroform solvent system. By 4 hours, the spotcorresponding to paclitaxel (R_(f) =0.3) was no longer evident; a spotnot present in the analysis of the initial reaction mixture (R_(f) =0.5)was present.

Water (25 ml) was added to the reaction, and then extracted intochloroform to remove most of the DMAP. After drying with magnesiumsulfate, material from the chloroform phase was dissolved in 1% methanolin chloroform. The dissolved material was then added to a plug of silicaGel 60 (Fluka Fine Chemicals) 4 cm high×4 cm diameter, and 300 ml of a1% methanol-in-chloroform mixture was run through the plug.

The resulting compound was identified as 2'caproyl-paclitaxel(paclitaxel in which CH₃ (CH₂)₄ C(O)-- was attached tothe 2'--OH group of pacitaxel) by NMR spectroscopy (see below).

7 lauroyl-paclitaxel

Paclitaxel (50 mg, 0.059 mmoles), DMAP (54 mg, 0.44 mmoles), lauroylchloride (77 mg, 0.35 mmoles), and 5 ml of chloroform were combined in a50 ml round-bottom flask, and incubated at room temperature. Reactionprogress was monitored as described above. At 4 hours of incubation, thespot corresponding to paclitaxel was no longer present, the spotcorresponding to 2' lauroyl-paclitaxel (R_(f) =0.5) was the largest, andanother spot (R_(f) =0.7) began to appear. At 24 hours, the spotcorresponding to 2' lauroyl-paclitaxel had disappeared, and the spot(R_(f) =0.7) corresponding to paclitaxel acetylated at both the 2' and 7positions had increased in size.

The reaction was then extracted, and run through a silica plug, asdescribed above. Flash chromatography, using a solvent system comprising1.5% methanol-in-chloroform was used to separate the material at spotR_(f) =0.7 from the material running with the solvent front. Thecompound was identified as 2',7 di-caprolypaclitaxel by NMRspectroscopy.

Fifty-eight mg (0.048 mmoles) of the diacetylated material was combinedwith 4.2 mg of sodium bicarbonate dissolved in 75 ul of water, in 30 mlof chloroform/methanol (1:1). Reaction progress was assayed frequently,so as to minimize hydrolysis of other ester linkages on the moleculewhile hydrolyzing the 12 carbon residue present at the 2' position. At 8hours, one major peak (R_(f) =0.5), and two minor peaks, (R_(f) =0.55,0.45), were observed; however, most of the material in the reactionmixture appeared to be starting material. Further incubation did notsubstantially increase the major spot (R_(f) =0.5). Accordingly, 25 mlof water was added to the reaction mixture, and then extracted intochloroform. Preparative TLC chromatography (Whatman 20×20 c, fluorescentat @ 254 nm, 1000 micron plate) was used to purify the compounds. Thecompound was identified as 7 caproyl-paclitaxel by NMR spectroscopy.

NMR Spectroscopy

¹ H and proton-decoupled ¹³ C spectra of paclitaxel reacted with 6, 12and 18 carbon fatty acids were taken. Shifts in the resonance identifiedas being protons alpha to the hydroxyl groups are indicative of the ofacylation of the corresponding hydroxyl group (see, for example,Kingston, Pharm. Ther. 52 (1991), pp. 1-34). Reactions with the 2' OHgroup proton are characterized by disappearance of resonance at 3.6 ppmand a shift of the proton alpha to the hydroxyl group from 4.8 ppm to5.6 ppm. Similar changes were reported by Kingston upon acetylation ofthe 2' OH group.

Paclitaxel derivatized with fatty acids at the 2' and 7 positions showthe same changes as occur with 2' derivatization, as well as loss ofresonance at 2.5 ppm (7 OH group proton) and a resonance shift from 4.5ppm to 5.65 ppm. Reactions with the 7 hydroxy proton are characterizeddisappearance of resonance at 2.5 ppm, and a shift of the proton alphato the OH group from 4.5 to 5.65 ppm. Similar to changes were reportedby Kingston with acetylation of the 7 OH group¹. Carbon spectra ofpaclitaxel attached to a fatty acid have an additional resonance, infrequencies associated with the carbonyl functional group (165 to 200ppm), as well as resonances in frequencies associated with aliphaticcarbons (10 to 60 ppm).

Example 2

Association Assays

Liposomes were prepared, using the lipid concentrations indicated in thetable below ("DSPC"=distearoyl phosphatidylcholine; "EPC"=eggphosphatidylcholine; "GA"=dipalmitoyl phosphatidylethanolamine/GlutaricAcid; "Chol"=cholesterol; "drug"=taxane derivatives), and using standardprocedures to make multilamellar liposomes; these were then extruded tentimes through a filter having 100-nm pores so as to obtain unilamellarliposomes (see Cullis et al., U.S. Pat. No. 5,008,050).

Paclitaxel and hydrophobic derivatives of paclitaxel were formulated inliposomes composed of POPC. 54.6 nanomoles of the liposomal formulationof each drug was passed through a 55 cm×3 cm column filled with BioGelA-15m. 200-400 mesh Bio-Rad agarose beads, at a flow rate of 6 cm/min.

Results are presented in FIG. 1. For paclitaxel approximately 20% of thedrug remains associated with liposomes after passage through the column.However, for 7 caprolylpaclitaxel approximately 90% of the drug remainsassociated with liposomes.

Table 1, the "Summary of the Formulation Study Table," presented belowis a summary of the results from several studies of the effect of theliposomal composition on 7 caproyl-paclitaxel and 7 stearoyl-paclitaxel.In particular, effects of saturation, cholesterol and PE-GA inclusionwere examined. Column 1 describes the liposomal formulation examined,column 2 the amount of drug associated with liposomes after passagethrough gel filtration columns (as described above), column 3 is aqualitative index of liposome aggregation (as observed by microscopy),and column 4 the mole percent of taxane associated with lipid afterliposome formation and extrusion through 100 nm filters. Unlessotherwise stated, initial paclitaxel concentration was 5 mole percent.

                  TABLE 1    ______________________________________    SUMMARY OF FORMULATION STUDY                 % RETAINED        % drug post    FORMULATION    Avg.   Std. Dev.                                   AGG.  extrusion    ______________________________________    Stearoyl-paclitaxel    DSPC; 10% GA; 5% Drug                   49      1       --    4    EPC; 10% GA; 5% Drug                   78     14       --    3    EPC; 25% CHOL; 5% Drug                   129    25       ***   4    7 caproyl-paclitaxel    EPC; 20% GA; 15% Drug                   56     10       --    12    EPC; 10% GA; 15%; Drug                   44      7       --    14    EPC; 10% GA; 5% Drug                   37     11       --    8    EPC; 25% CHOL; 5% Drug                   31      1       **    6    EPC; 25% CHOL; 10% GA;                   43     11       *     6    5% Drug    DSPC; 10% GA; 5% Drug                   61     20       --    3    DSPC; 25% CHOL; 5% Drug                   45              ****  1    DSPC; 25% CHOL, 10% GA;                   30              **    1    5% Drug    ______________________________________

Example 3

Characterization of Taxane-Containing POPC Liposomes

Palmitoyloleoyl phosphatidylcholine (POPC) liposomes containing eitherpaclitaxel itself or a taxane-hydrophobic organic moiety conjugate wereprepared at a mole ratio of 95:5, lipid:drug according to proceduresdescribed hereinabove. The lipid, conjugate and paclitaxelconcentrations were determined after liposome preparation, as well asbefore and after passing through a size exclusion column (200-400 meshBio-Rad agarose beads). The decrease, if any, in the association ofpaclitaxel or the conjugate with the POPC was calculated and used as ameasure of association with liposomes. Results are presented in Table 2,below.

                  TABLE 2    ______________________________________    LIPOSOME ASSOCIATION STUDIES    Compound      % Taxane Associated with Liposomes    ______________________________________    2'-caproyl paclitaxel                  40.8    2'-lauroyl paclitaxel                  50.9    2'-stearoyl paclitaxel                  65.7    7-caproyl paclitaxel                  63.2    7-lauroyl paclitaxel                  72.6    7-stearoyl paclitaxel                  89.5    Paclitaxel    21.7    ______________________________________

Example 4

In Vivo Studies

Liposomes were made, with dipalmitoyl phosphatidylcholine (DSPC),distearoyl phosphatidylethanolamine-glutaric acid (DPPE-GA) and7-caproyl paclitaxel (7-C6), according to procedures describedhereinabove at a molar ratio of DSPC:DPPE-GA:7-C6 of 8:1:0.6.

P-388 Studies

Groups of mice were injected with 1×10⁵ P388 cells and then, 24 hourslater, either Taxol® (cremophor-based paclitaxel suspension) or ataxane-containing liposome. "Plain," that is, non-taxane-containing,liposomes were administered at 512 mg/kg of body weight, which wasequivalent to a 50 mg/kg dose of the taxane-containing liposomes.Liposome, or Taxol®, administration was repeated at days 2, 3, 4 and 5post cell administration. Days of death were then observed, and meansurvival times calculated. These data are presented in Table 3, below.

                  TABLE 3    ______________________________________    SURVIVAL OF P388 TUMOR-BEARING MICE                         Mean Survival Time,                                       ILS.sup.1 as of    Treatment Dose (mg/kg)                         Days (# mice) Control    ______________________________________    PBS.sup.2 0.2    ml      11.6 ± 0.2 (5)                                         --    Plain Liposomes              512    mg/kg   11.2 ± 0.2 (5)                                         (-)3.5 .sup.    7-C6 Liposomes              50             17.2 ± 0.7 (5)                                         48.2    7-C6 Liposomes              25              13.2 ± 0.2 (10)                                         13.8    Taxol ®              12.5            17.6 ± 0.8 (10)                                         51.7    ______________________________________     .sup.1 "ILS" = increased life span = ((mean survival time (MST)treatment     group/MST control) × 100) - 100;     .sup.2 phosphate buffered saline.

B-16 Studies

Groups of mice were injected (intravenously) with 1×10⁵ B-16 cells, andthen, one day later, either PBS, plain liposomes, Taxole, or 7-C6liposomes. Treatment was repeated on days 3, 5, 7 and 9 postcelladministration. On day 21, the animals were sacrificed, and their lungswere removed and fixed in formalin. The number of melanotic lung noduleswas counted "blind" using a magnifier; data is presented in Table 4,below.

                  TABLE 4    ______________________________________    EFFECT ON DEVELOPMENT OF B16 MELANOMA LUNG TUMORS                         Mean Number of                                     % Reduction in                         Tumor Nodules                                     Nodule # vs.    Treatment Dose (mg/kg)                         (# mice)    PBS-Control    ______________________________________    PBS.sup.2 0.2    ml      27.1 ± 2.6 (8)                                       --    Plain Liposomes              512    mg/kg   28.6 ± 1.4 (8)                                       (-)5.5 .sup.    7-C6 Liposomes              50             11.4 ± 1.2 (8)                                       57.9    7-C6 Liposomes              25             13.3 ± 2.3 (8)                                       50.9    Taxol ®              12.5           17.5 ± 2.7 (8)                                       35.4    ______________________________________

Example 5

Growth Inhibition Studies

Cells (A549, MCF7 or Lewis Lung) were incubated with variousconcentrations of paclitaxel; cell growth inhibition was determined bystandard means. Results of these experiments are presented in Tables 5and 6 (below), with the data there indicating the concentration (GI₅₀),micromolar, of either free paclitaxel, or a free paclitaxel derivative(Table 5) or liposomal paclitaxel or derivative (Table 6), that is, theconcentration found to inhibit about fifty percent of the growth of theindicated cell lines.

                  TABLE 5    ______________________________________    GROWTH INHIBITION BY THE PACLITAXEL AND    IT'S DERIVATIVES                Cell Line             LEWIS    Compound    A549       MCF7       LUNG    ______________________________________    Paclitaxel  0.004 +/-  0.004 +/-  0.031 +/-                0.0001     0.0001     0.012    2'-C6       0.41 +/-   0.50 +/-   1.22 +/-                0.134      0.151      0.592    2'-C12      0.45 +/-   0.84 +/-   1.26 +/-                0.08       0.17       0.87    2'-C18      7.56 +/-   >6.0       >10                1.513    2',7-diC6   >10        >10        >10    2',7-diC12  >10        >10        >10    2',7-diC18  --*        --         --    7-C6        0.032 +/-  0.027 +/-  0.091 +/-                0.002      0.019      0.019    7-C12       4.71 +/-   >10        7.89 +/-                0.25                  0.37    7-C18       >10        >10        >10    ______________________________________     *Not soluble in DMSO or EtOH.

                  TABLE 6    ______________________________________    GROWTH INHIBITION BY LIPOSOMAL (POPC) PACLITAXELS                                      LEWIS    Taxane      A549       MCF7       LUNG    ______________________________________    Paclitaxel  0.002 +/-  <0.003     0.04 +/-                0.001                 0.03    2'-C6       0.40 +/-   0.22 +/-   0.86 +/-                0.01       0.01       0.15    2'-C12      0.37 +/-   0.19 +/-   1.68 +/-                0.02       0.01       0.70    2'-C18      3.70 +/-   1.21 +/-   >10                0.28       0.06    2',7-diC6   >10        >10        >10    2',7-diC12  >10        >10        >10    2',7-diC18  >10        >10        >10    7-C6        0.031 +/-  0.018 +/-  0.071 +/-                0.001      0.003      0.001    7-C12       4.36 +/-   4.79 +/-   >10                0.14       4.85    7-C18       >10        >10        >10    ______________________________________

What is claimed is:
 1. A method of preparing a compound comprising ataxane and a hydrocarbon chain attached to the hydroxyl group at theposition 2' of the taxane, said method comprising the step of using astoichiometric amount of an active form of the hydrocarbon chain toattach the chain to the taxane;wherein the active form of thehydrocarbon chain is a chloride or anhydride; wherein the attachment isin the presence of: (i) a base selected from the group consisting ofpyridine, dimethylaminopyridine and triethylamine: and, (ii) a polar,aprotic solvent; wherein the taxane prepared by said method has theformula: ##STR12## and wherein: A¹ is a group having the formulaQ--C(O)NHCH(C₆ H₅)CH(OR)C(O)--; Q is C₆ H₅ --, (CH₃)₃ C--O-- or(CH₃)CH═C(CH₃)--; A² is H or CH₃ C(O)--; R is a hydrocarbon chain havingthe formula --C(O)(CH₂)_(a) (CH═CH)_(b) (CH₂)_(c) (CH═CH)_(d) (CH₂)_(e)(CH═CH)_(f) (CH₂)_(g) (CH═CH)_(h) (CH₂)_(i) CH₃, the sum ofa+2b+c+2d+e+2f+g+2h+i is equal to an integer of from 7 to 22, a is equalto zero or an integer from 1 to 22, each of b, d, f and h isindependently equal to zero or 1, c is equal to zero or an integer from1 to 19, e is equal to zero or an integer from 1 to 16, g is equal tozero or an integer from 1 to 13, i is equal to zero or an integer from 1to 10, and a to i can be the same or different at each occurrence. 2.The method of claim 1, wherein A² is CH₃ C(O)--.
 3. The method of claim2, wherein A¹ is a group having the formula Q--C(O)NHCH(C₆H₅)CH(OR)C(O)--.
 4. The method of claim 3, wherein Q is C₆ H₅ --.
 5. Themethod of claim 4, wherein R¹ is the group --C(O)(CH₂)_(a) (CH₃.
 6. Themethod of claim 5, wherein R¹ is --C(O)(CH₂) ₁₀ CH₃ or --C(O)(CH₂)₁₆CH₃.
 7. A method of attaching an additional hydrocarbon chain to thetaxane produced by the method of claim 1, wherein:said additionalhydrocarbon chain is attached to the hydroxyl group at the 7 position ofthe taxane; said additional hydrocarbon chain is the same as thehydrocarbon chain attached to the hydroxyl group at the 2' position ofthe taxane; and, said method comprises the step of reacting the taxanewith an amount of the reactive form of the hydrocarbon chain which isgreater than a stoichiomteric amount of the chain.
 8. The method ofclaim 7 comprising the additional step of removing the hydrocarbon chainattached to the taxane at the 2' position so as to Produce a taxanehaving a hydrocarbon chain attached only to the 7 position, said methodcomprising the step of reacting the taxane with a stoichiometric amountof a mild base.
 9. A method of preparing a compound comprising a taxaneand a hydrocarbon chain attached to the hydroxyl group at the 7 positionof the taxane, said method comprising the steps of:(a) reacting thetaxane with a moiety selected from the group consisting of triphenylmethyl, methoxytriphenyl methyl, trifluoroacetyl and hexanoyl moietiesso as to attach the moiety to the hydroxyl group at the 2' position ofthe taxane; (b) reacting the taxane of step (a) with a chloride oranhydride form of the hydrocarbon chain so as to attach the chain to thehydroxyl group at the 7 position of the taxane but not to the hydroxylgroup at the 2' position, wherein said reaction is conducted using:(i) abase selected from the group consisting of pyridine,dimethylaminopyridine and triethylamine; and, (ii) a polar, agroticsolvent; and, (c) removing the moiety attached to the 2' position of thetaxane, wherein the taxane produced by said method has the formula:##STR13## and wherein: A¹ is a group having the formula Q--C(O)NHCH(C₆H₅)CH(OH)C(O)--; Q is C₆ H₅ --, (CH₃)₃ C--O-- or (CH₃)CH═C(CH₃)--, A² isH or CH₃ C(O)--; R¹ is a hydrocarbon chain having the formula--C(O)(CH₂)_(a) (CH═CH)_(b) (CH₂)_(c) (CH═CH)_(d) (CH₂)_(e) (CH═CH)_(f)(CH₂)_(g) (CH═CH)_(h) (CH₂)_(i) CH₃, the sum of a+2b+c+2d+e+2f+g+2h+i isequal to an integer of from 7 to 22, a is equal to zero or an integerfrom 1 to 22, each of b, d, f and h is independently equal to zero or 1,c is equal to zero or an integer from 1 to 19, e is equal to zero or aninteger from 1 to 16, g is equal to zero or an integer from 1 to 13, iis equal to zero or an integer from 1 to 10, and a to i can be the sameor different at each occurrence.
 10. The method of claim 9, wherein A²is CH₃ C(O)--.
 11. The method of claim 10, wherein A¹ is a group havingthe formula Q--C(O)NHCH(C₆ H₅)CH(OH)C(O)--.
 12. The method of claim 11,wherein Q is C₆ H₅ --.
 13. The method of claim 12, wherein R¹ is--C(O)(CH₂)_(a) CH₃.
 14. The method of claim 13, wherein R¹ is--C(O)(CH₂)₁₀ CH₃ or --C(O)(CH₂)₁₆ CH₃.